(a) Field of the Invention
The invention relates to the use of nucleotide substitutes for increasing the in vivo efficacy of nucleic acid molecules and also for inhibiting inflammation in mammals.
More particularly, the present invention relates to the use of 2′6′diaminopurine (DAP) and analogs thereof per se in anti-inflammatory compositions, and also for preparing nucleic acid molecules having an increased in vivo physiological efficiency and a reduced toxicity.
(b) Brief Description of the Prior Art
Therapeutic approaches based on the use of nucleic acid molecules are becoming more and more popular. Gene-based therapies and antisense based-therapies will probably change radically medicine in a near future.
The problems to date with nucleic acid molecules as therapeutics, and more particularly with antisense oligonucleotides, have been toxicity (both systemic and topical), stability, and non-specific binding to cell surface proteins. The toxicity of antisense oligonucleotides seems to vary between species, rats being the most sensitive, although the toxicity appears at doses higher than those that are therapeutically effective (see S T Crooke, Hematologic Pathology, 1995, 9:5972 for a review). In pulmonary/respiratory diseases, nucleic acid molecules toxicity associated with the administration of therapeutic antisenses/genes include: an increase in immune stimulation, a mononuclear cellular-inflammatory infiltrate into the lungs, and possibly hypersensitivity and bronchoconstriction of the airways.
Several solutions that are less than optimum have been proposed up to date for circumventing the toxicity problem. Among the most popular there is the preparation of nucleic acid molecules containing various modified DNA bases, RNA bases, and/or a modified backbone structure. For instance, WO 99/67378 describes antisense oligonucleotides constructs based on modified sugars. Also, Nyce has postulated, although not demonstrated, in WO 00/09525 and WO 00/62736 that the adenosine base included in antisense oligonucleotides for treating respiratory diseases is a major cause of toxicity in lungs. Accordingly, Nyce proposes low adenosine oligonucleotides and oligonucleotides wherein the adenosine base has been replaced by an analog of adenosine. However, none of the low adenosine oligonucleotides and none of the adenosine analogs proposed by Nyce have ever been tested for their biological activity or their allegedly reduced toxicity.
2′,6′-diaminopurine nucleoside (2-amino-2′-deoxyadenosine; DAP) was found to be present in DNA in place of adenosine by the cyanophage S-2L (Cheng, X., Annu Rev Biophys Biomol Struct 24: 293-318, 1995); Khudyakov, I. Y., et al., Virology 88: 8-18, 1978). Since then, 2′,6′-diaminopurine nucleoside (DAP) has been widely used and studied, notably as a chemical starting point for the synthesis of antiviral compounds such as 2-amino-2′,3′-dideoxyadenosine (not DAP) which is capable of selectively inhibiting human immunodeficiency virus (HIV) replication in vitro (Balzarini, J. et al., Biochem. & Biophys. Res. Communications 145:269-76 (1987). The use of DAP in antisense oligonucleotides or in gene therapy methods has however never been suggested.
Also, U.S. Pat. No. 5,925,624 and No. 5,889,178 describe derivatives of 2,6-diaminopurine-beta-D-ribofuranuronamide. Although these derivatives have an anti-inflammatory effect (mostly against neutrophil superoxide release) and that they could be used in the therapy of respiratory disease, they have a chemical formula which is different from the formula of DAP and analogs thereof.
In summary, there has been up to date no suggestion nor any evidence that DAP per se could be used in anti-inflammatory compositions, nor any suggestion or example that DAP and analogs thereof could be incorporated in nucleic acid molecules (gene constructs and antisenses) for increasing the in vivo efficacy of these oligos.
There is thus a need for more effective anti-inflammatory compositions comprising 2′6-diaminopurine and/or analogs thereof.
There is also a long felt need for nucleic acid molecules that would remain stable in the body while exhibiting high effectiveness and low toxicity.
There is more particularly a need for nucleic acid molecules incorporating a nucleotide substitute such as 2′6′diaminopurine (DAP) and analogs thereof, a need for composition comprising the same and a need for methods of using these nucleic acid molecules, particularly in gene and antisense therapies methods. No one has ever tested whether replacement of base(s) by a nucleotide substitute could affect the stability, binding, degradation efficacy and toxicity of antisense oligonucleotides, nor have they tested such modified antisense oligonucleotides for biological activity in cells, in culture or in animals.
The present invention fulfils these needs and also other needs which will be apparent to those skilled in the art upon reading the following specification.